Periodic Reporting for period 1 - GlycoNanoPep (Glyco and Nano Peptide Conjugates for Selective Cell Penetration)
Période du rapport: 2017-04-01 au 2019-03-31
Recent advances in genomic technologies have made possible the identification of new therapeutic targets that could be addressed with a variety of drugs (small molecules, proteins) or gene therapies (DNA, siRNA, CRISPR). Although this wide range of potential drugs is available, their use is however hampered by the intrinsic difficulty in the delivery of drug and genes into the cells of interest. In addition, further difficulties arise from the interaction of the cargos (i.e. nucleotides) with the diverse components of the biological media, which includes the presence of nucleases, proteases and other dissolved proteins. Therefore, the design of innovative delivery systems that can protect the drug and improve their selective and efficient biodistribution in a complex biological media constitutes a critical stepping-stone towards the development of new nanomedicines or imaging strategies. The goal of this project is the development of dynamic multivalent glycoconjugated cell penetrating peptides (DyGCPPs) and nanoparticles (GNP-DyCPPs) as a powerful tool for selective membrane permeation of active components at a cellular level. This might serve for the development of new therapeutic strategies that prevent side effects of the conventional biodistribution cell penetrating peptides (CPP): allow specific delivery of CPP to specific tissues and prevent degradation of CPPs by interaction with biological components (serum, proteins, etc.). The work developed in this proposal has allowed the control over the formation of supramolecular nanoparticles constituted by multiple elements and their intracellular delivery using penetrating peptide vehicles. These results will open new directions for conceptually new artificial drug delivery systems. This new knowledge will benefit the progress in key enabling technologies for the future social challenges in health and biomedicine.
- Integration of cell penetrating peptides into gold nanoparticles by host-guest interactions: We have prepared gold nanoparticles bearing host anchored to the surface (beta-cyclodextrins). We have synthesized monovalent and divalent guest-bearing cell penetrating peptides. By combination of the building blocks, we have developed multicomponent nanoparticles bearing different surface coverage of peptide and stabilizers (PEGs). Their properties and morphology was studied by several techniques (DLS, TEM, zeta potential measurements, UV). We have observed that the binding of the CPP to guest nanoparticles have a mayor host-guest contribution and a minor electrostatic contribution. In this way, we were able to avoid nanoparticle aggregation by using statistical/multivalent interactions.
The following items indicate concrete points developed during the grant:
• Preparation of host-bearing cell penetrating peptides: We have developed efficient synthetic strategies towards the preparation of a variety of cell penetrating peptides that include several functionalities (e.g. guest motives, fluorophores anchored). This has been accomplished by synthetic strategies including solid phase and homogeneous solution synthesis.
• Demonstration of the functionalization of CPPs in nanoparticles by host-guest chemistry, including the use of multivalent interactions, and demonstrate their release: We have demonstrated the formation of supramolecular host-guest complexes between β -CD@AuNPs and adamantane-peptides and adamantane-PEG by several techniques, such as DLS, zeta-potential measurements and fluorescence measurements. The release of the cargo has been demonstrated with competition experiments. We also have studied the importance of the molecular design towards multivalent recognition between nanoparticles and peptides.
• Determination of the critical parameters for the stabilization of nanoparticles in different conditions: We carried out a detailed investigation concerning the the stability of the assemblies in conditions which are biologically relevant. Overall, we have observed a more important stabilizing effect due to electrostatic factors rather than by steric stabilization (e.g. like the stabilization achieved by adding polyethyleneglycol stabilizers).
• Obtaining information about binding to cell membranes: We have evaluated the interaction between CPP anchored to nanoparticles in cell models (HeLa) and demonstrated efficient uptake and cytosolic delivery of the peptide after host/guest release.
- Peptide exchange within cells: We have further evaluated the dynamic exchange of peptides within cells, for this we have developed nanoparticles loaded with one cell type that were delivered into cells. The addition of a second peptide that penetrated the cells and reached the nanoparticle allowed the exchaThe results of this propoposal have already given rise to two publications up to date and we have attended to two international symposiums to present these results (including one poster prize). Aditionally, three more maniscripts are currently being prepared with the most promising results in cells and delivery that will be pu blished in the best multidisciplinary chemistry journals.nge of one peptide for another. This could be employed for therapeutic strategies.
The following items indicate concrete points developed during the grant:
• Preparation of host-bearing cell penetrating peptides: We have developed efficient synthetic strategies towards the preparation of a variety of cell penetrating peptides that include several functionalities (e.g. guest motives, fluorophores anchored). This has been accomplished by synthetic strategies including solid phase and homogeneous solution synthesis.
• Demonstration of the functionalization of CPPs in nanoparticles by host-guest chemistry, including the use of multivalent interactions, and demonstrate their release: We have demonstrated the formation of supramolecular host-guest complexes between β -CD@AuNPs and adamantane-peptides and adamantane-PEG by several techniques, such as DLS, zeta-potential measurements and fluorescence measurements. The release of the cargo has been demonstrated with competition experiments. We also have studied the importance of the molecular design towards multivalent recognition between nanoparticles and peptides.
• Determination of the critical parameters for the stabilization of nanoparticles in different conditions: We carried out a detailed investigation concerning the the stability of the assemblies in conditions which are biologically relevant. Overall, we have observed a more important stabilizing effect due to electrostatic factors rather than by steric stabilization (e.g. like the stabilization achieved by adding polyethyleneglycol stabilizers).
• Obtaining information about binding to cell membranes: We have evaluated the interaction between CPP anchored to nanoparticles in cell models (HeLa) and demonstrated efficient uptake and cytosolic delivery of the peptide after host/guest release.
- Peptide exchange within cells: We have further evaluated the dynamic exchange of peptides within cells, for this we have developed nanoparticles loaded with one cell type that were delivered into cells. The addition of a second peptide that penetrated the cells and reached the nanoparticle allowed the exchaThe results of this propoposal have already given rise to two publications up to date and we have attended to two international symposiums to present these results (including one poster prize). Aditionally, three more maniscripts are currently being prepared with the most promising results in cells and delivery that will be pu blished in the best multidisciplinary chemistry journals.nge of one peptide for another. This could be employed for therapeutic strategies.
We have demonstrated the supramolecular incorporation of CPP into β-CD@AuNPs to give rise to new multicomponent systems where the nanoparticles properties can be easily tuned. We have thorougly characterized the interplay between assembly and stability of nanoparticles, and developed multivalency-based strategies towards efficient incorporation of CPP into host nanoparticles. We have demonstrated also complexation-dependend cell uptake of nanoparticles of negatively charged peptides. We have make use of competition between monovalent and multivalent interactions to trigger cell release of independent peptides. This will be instrumental to develop new therapeutic strategies based on affinity-driven host-guest exchange within live cells.
Particular objectives achieved beyond the state of the art are:
1. We have developed synthetic procedures for the preparation of cell penetrating peptides bearing multiple functional elements (fluorophores, guest moieties).
2. We have demonstrated the formation of supramolecular complexes between β -CD@AuNPs and guest-bearing cell penetrating peptides. Furthermore, we have shown that the inclusion of monovalent and multi-
valent motives can be used to modulate the binding affinity between these two elements.
3. We have carried out an exhaustive analysis of the formation of supramolecular nanoparticles constituted by monovalent/divalent cell penetrating peptides, monovalent/divalent polymeric stabilizers and β -CD@AuNPs.
We have thoroughly characterized their physicochemical properties (size, surface charge) as well as their stability towards several relevant biological conditions.
4. In connection to the 3rd point, we have shown that the stabilization of these supramolecular nanoparticles is mostly electrostatic, while steric stabilization by including pendant hydrophilic polymers had a minor effect.
5. We have demonstrated host-guest exchange within live cells mediated by competition between monovalent and divalent interactions, and analyzed the cellular localization of the process.
Particular objectives achieved beyond the state of the art are:
1. We have developed synthetic procedures for the preparation of cell penetrating peptides bearing multiple functional elements (fluorophores, guest moieties).
2. We have demonstrated the formation of supramolecular complexes between β -CD@AuNPs and guest-bearing cell penetrating peptides. Furthermore, we have shown that the inclusion of monovalent and multi-
valent motives can be used to modulate the binding affinity between these two elements.
3. We have carried out an exhaustive analysis of the formation of supramolecular nanoparticles constituted by monovalent/divalent cell penetrating peptides, monovalent/divalent polymeric stabilizers and β -CD@AuNPs.
We have thoroughly characterized their physicochemical properties (size, surface charge) as well as their stability towards several relevant biological conditions.
4. In connection to the 3rd point, we have shown that the stabilization of these supramolecular nanoparticles is mostly electrostatic, while steric stabilization by including pendant hydrophilic polymers had a minor effect.
5. We have demonstrated host-guest exchange within live cells mediated by competition between monovalent and divalent interactions, and analyzed the cellular localization of the process.